In vitro characterisation of J591C diabody. (a) SDS-PAGE of purified J591C diabody. The reduced sample runs as a single band corresponding to the size of the monomer (27 kDa). In the non-reduced sample, the majority of protein is present as a covalent dimer through formation of a disulfide bond by the C-terminal cysteines. (b) Analytical size exclusion HPLC. The UV chromatogram shows elution as a single dimeric species. (c) Saturation binding assay on DU145-PSMA and LNCaP cells with J591Cdia-AlexaFluor-488, labelled via the C-terminal cysteines and analysed by flow cytometry. DU145-PSMA cells show higher PSMA expression levels than LNCaP. (d) Binding and internalisation studies with the AlexaFluor-488-labelled diabody in DU145-PSMA and DU145 cells at 4°C and 37°C (20 min of incubation). (e) Competitive binding assay with 1 nM [99mTc(CO)3]+-labelled diabody vs. unlabelled diabody as cold competitor on DU145-PSMA and DU145 cells (data representative of four independent experiments).